The present invention relates to the use of a 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine derivative and physiologically functional derivatives thereof for the treatment of hepatitis B viral infections.
Hepatitis B virus (HBV) is a viral pathogen of major worldwide importance. HBV is most common in Asian Countries, and prevalent in sub-Saharan Africa. The virus is etiologically associated with primary hepatocellular carcinoma and is thought to cause 80% of the world""s liver cancer. In the United States more than ten thousand people are hospitalized for HBV illness each year, an average of 250 die with fulminant disease. The United States currently contains an estimated pool of 500,000-1 million infectious carriers. Chronic active hepatitis will develop in over 25% of carriers and often progresses to cirrhosis. It is estimated that 5000 people die from HBV-related cirrhosis each year in the U.S.A. and that perhaps 1000 die from HBV-related liver cancer. Even when a universal HBV vaccine is in place, the need for effective anti-HBV compounds will continue. The large reservoir of persistently infected carriers, estimated at 220 million worldwide, will receive no benefit from vaccination and will continue at high risk for HBV-induced liver disease. This carrier population serves as the source of infection of susceptible individuals perpetuating the instance of disease particularly in endemic areas or high risk groups such as i.v. drug abusers and homosexuals. Thus, there is a great need for effective antiviral agents, both to control the chronic infection and reduce progression to hepatocellular carcinoma.
Clinical effects of infection with HBV range from headache, fever, malaise, nausea, vomiting, anorexia and abdominal pains. Replication of the virus is usually controlled by the immune response, with a course of recovery lasting weeks or months in humans, but infection may be more severe leading to persistent chronic liver disease as outlined above. In xe2x80x9cViral Infections of Humansxe2x80x9d (second edition, Ed. Evans, A. S. (1982) Plenum Publishing Corporation, New York), Chapter 12 describes the etiology of viral hepatitis infections.
European Patent Specification 0 382 526 discloses certain 1,3-oxathiolane nucleoside analogues which are effective in inhibiting the replication of human immunodeficiency virus (HIV).
Since the priority date of this patent application, the following items have been published: Doong, S. L. et al., Inhibition of the replication of hepatitis B virus in vitro by 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-thiacytidine and related analogs, Proc. Natl. Acad. Sci. USA, 88 (19), 8495-9 (1991); Liotta, D. C. and Choi, W. B., Synthesis of BCH-189 and related compounds, PCT Appl. WO 91/11186; Soudeyns, H. et al., Anti-human immunodeficiency virus type 1 activity and in vitro toxicity of 2xe2x80x2-deoxy-3xe2x80x2-thiacytidine (BCH-189), Antimicrob. Agents Chemother., 35 (7), 1386-90 (1991) and Choi, W. B. et al., In situ complexation directs the stereochemistry of N-glycosylation in the synthesis of thialanyl and dioxolanyl nucleoside analogs, J. Am. Chem. Soc., 113 (24), 9377-9 (1991).
We have now surprisingly found that a 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine derivative of formula I 
namely 1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine or a pharmaceutically acceptable salt, ester or other physiologically functional derivatives thereof have potent activity against HBV.
It should be noted that the compound of formula (I) contains two chiral centers and therefore exists in the form of two pairs of optical isomers (i.e. enantiomers) and mixtures thereof including racemic mixtures. Thus, the compound of formula (I) may be either a cis or a trans isomer or mixtures thereof. Each cis and trans isomer can exist as one of two enantiomers or mixtures thereof including racemic mixtures. All such isomers and mixtures thereof including racemic mixtures and tautomeric forms of the compound of formula (I) are within the scope of the invention. The cis isomers of the compound of formula (I) are preferred.
According to one feature of the present invention we provide the compound of formula (I) or a physiologically functional derivative thereof for use in the treatment or prophylaxis of a hepatitis B virus infection. According to a further feature of the present invention we provide the use of the compound of formula (I) or a physiologically functional derivative thereof, in the manufacture of a medicament for the treatment or prophylaxis of a hepatitis B virus infection.
In a further aspect of the present invention there is included a method for the treatment or prophylaxis of a hepatitis B virus infection in a host, for example, a mammal such as a human which comprises treating the host with a therapeutically effective amount of the compound of formula (I) or a physiologically functional derivative thereof.
By xe2x80x9cphysiologically functional derivativexe2x80x9d is meant a pharmaceutically acceptable salt, amide, ester or salt of an ester of the compound of formula (I) or any other compound which upon administration to the recipient, is capable of providing (directly or indirectly) the said compound of formula (I) or an active metabolite or residue thereof.
Preferred esters in accordance with the invention include carboxylic acid esters in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl e.g. n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), arylalkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), and aryl (e.g. phenyl); sulfonate esters such as alkyl- or arylalkylsulfonyl (e.g. methanesulfonyl); amino acid esters (e.g. L-valyl or L-isoleucyl); dicarboxylic acid esters (e.g. hemisuccinate); and 5xe2x80x2- mono- di- or tri-phosphate esters. The phosphate esters may be further esterified by, for example, a C1-20 alcohol or reactive derivative thereof, or by a 2,3-di(C6-24)acyl glycerol. Any alkyl moiety present in such esters advantageously contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group optionally substituted e.g. by halogen, C1-4 alkyl, C1-4 alkoxy or nitro.
The above-mentioned pharmaceutically acceptable amides of the compound of formula (I) include those derivatives wherein the cytosine amino group is present in the form of an amide, e.g. xe2x80x94NHCOR where R is C1-6 alkyl or aryl (e.g. phenyl optionally substituted by halogen, C1-4 alkyl, C1-4 alkoxy, nitro or hydroxyl).
Examples of pharmaceutically acceptable salts according to the invention include base salts, e.g. derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NX4+ (wherein X is C1-4 alkyl). Pharmaceutically acceptable acid addition salts include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
The amount of the compound of formula (I) (hereinafter also referred to as the xe2x80x9cactive ingredientxe2x80x9d) or physiologically functional derivative thereof which is required in a medication to achieve the desired effect will depend on a number of factors, in particular the specific application, the nature of the particular compound used, the mode of administration and the condition of the patient. In general a suitable dose will be in the range of 3.0 to 120 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg per kilogram body weight per day and most preferably in the range 15 to 60 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form.
Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active ingredient of from about 1 to about 75 xcexcM, preferably about 2 to 50 xcexcM, most preferably about 3 to about 30 xcexcM. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to about 100 mg/kg of the active ingredient. Desirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg/kg of the active ingredient.
In the manufacture of a medicament according to the invention, hereinafter referred to as a xe2x80x9cformulationxe2x80x9d, the compound of formula (I) or a physiologically functional derivative thereof herein as xe2x80x9cactive ingredientxe2x80x9d, is typically admixed with, inter alia, one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents.
The formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
Formulations suitable for oral use as described above may also include buffering agents designed to neutralize stomach acidity. Such buffers may be chosen from a variety of organic or inorganic agents such as weak acids or bases admixed with their conjugated salts.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound 1) in an optionally buffered, aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3) dispersed in a polymer. A suitable concentration of the active compound is about 1% to 35%, preferably about 3% to 15%. As one particular possibility, the active compound may be delivered from the patch by electrotransport or iontophoresis as generally described in Pharmaceutical Research, 3 (6), 318 (1986).
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injections solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, as liposomes or other microparticulate systems which are designed to target the compounds to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
The compound of formula I may be prepared for example by:
a) reacting an optionally protected 5-F-cytosine compound with a 1,3-oxathiolane of formula (IIA) 
wherein R1 is hydrogen or a hydroxy protecting group and L is a leaving group; or
b) reacting a compound of formula (IIB) 
(wherein R1 is as defined above and R1a is an amino protecting group) with a fluorinating agent serving to introduce a fluorine atom in the 5-position of the cytosine ring; or
c) reacting a compound of formula (IIC) 
(wherein R1 is as defined above) with an agent serving to convert the oxo group in the 4-position of the uracil ring to an amino group; any remaining protecting groups being removed for example by acid or base hydrolysis to produce the desired product.
With regard to process a), the hydroxy protecting group includes protecting groups such as acyl (e.g. acetyl), arylacyl (e.g. benzoyl or substituted benzoyl), trityl or monomethoxytrityl, benzyl or substituted benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. The 5-F-cytosine compound may be optionally protected with silyl, e.g. trimethyl silyl groups. Such groups may be removed in conventional manner. The leaving group L is a leaving group typical of those known in the art of nucleoside chemistry e.g. halogen such as chlorine or bromine, alkoxy such as methoxy or ethoxy or acyl such as acetyl or benzoyl.
The reaction in process a) may be effected in an organic solvent (e.g. 1,2-dichloroethane or acetonitrile) in the presence of a Levis acid such as stannic chloride or trimethylsilyl triflate.
Compounds of formula IIA may be obtained from a suitably protected 2-hydroxyacetaldehyde of formula (III),
R1OCH2CHOxe2x80x83xe2x80x83(III)
wherein R1 is defined above, as described in Can. J. Research, 8, 129 (1933) and European Patent Specification 0 382 526. Reaction of compounds of formula (III) with a mercaptoacetal HSCH2CH(OR)2, wherein R is C1-4 alkoxy such as HSCH2CH(OC2H5)2, known in the art (Chem. Ber. 85:924-932, 1952), yields compounds of formula IIA wherein L is OR (alkoxy) e.g. methoxy or ethoxy. Alternatively, compounds of formula IIA, wherein L is alkoxy, may be converted to compounds of formula IIA wherein L is halogen or acyl by methods known in the art of carbohydrate chemistry.
Compounds of formula (III) may be prepared from 1,2-O-isopropylidene glycerol by introduction of R1 (e.g. trisubstituted silyl, benzyl or trityl) and removal of the isopropylidene group with mild acid (e.g. aqueous formic or acetic acid) or zinc bromide in acetonitrile, followed by oxidation of the alcohol group with aqueous periodate.
With regard to process b), the 5-fluoro substituent may be introduced by methods known in the art (M. J. Robins, et al., in Nucleic Acid Chemistry, Part 2, L. B. Townsend and R. S. Tipson, editors, J. Wiley and Sons, New York, 895-900 (1978) and references therein; R. Duschinsky in Nucleic Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson, editors, J. Wiley and Sons, New York, 43-46 (1978) and references therein). The fluorinating agent may be, for example, trimethylhypofluorite in fluorotrichloromethane.
With regard to process c), the compound of formula (IIC) is advantageously treated with 1,2,4-triazole, advantageously together with 4-chlorophenyl dichlorophosphate, to form the corresponding 4-(1,2,4-triazolyl) compound which is then converted to the desired 4-amino (cytidine) compound by reaction with for example methanol.
The starting materials of formula (IIB) and (IIC) may be prepared for example by reaction of an appropriate (optionally protected) base with a compound of formula IIA in an analogous manner to that described in process a). 5-Fluorouracil and 5-fluorocytosine are commercially available from Aldrich Chemical Co., Milwaukee, Wis. 53233, USA.
Separation of the (xc2x1)-cis and (xc2x1)-trans isomers for example in a protected form, may be accomplished by chromatography on silica gel with mixtures of organic solvents such as ethyl acetate/methanol, ethylacetate/hexane or dichloromethane/methanol. Any protecting group may then be removed using the appropriate reagent for each group.
The compound of formula (I) may be converted into a pharmaceutically acceptable esters and amides by reaction with an appropriate acylating agent, for example, an acid halide or anhydride serving to acylate the 5xe2x80x2xe2x80x94OH and 4xe2x80x94NH2 groups. The acyl group may then be removed selectively from one or other of the 5xe2x80x2xe2x80x94OH and 4xe2x80x94NH2 groups. For, example, treatment of the diacylated compound under acidic conditions, eg. a Lewis acid such as zinc bromide in methanol, removes the 4N-acyl group to yield the corresponding 5xe2x80x2xe2x80x94OH ester where treatment of the diacylated compound under alkaline conditions, eg. with sodium methoxide removes the 5xe2x80x2xe2x80x94OH acyl group to yield the corresponding 4N-amide. The acyl groups can also be removed selectively by treatment with commercially available esterase or lipase enzymes, eg. pig liver esterase or pancreatic lipase, or by treatment in accordance with methods described in U.S. Pat. No. 5,071,983. The compound of formula (I) may be converted into a pharmaceutically acceptable salt thereof in a conventional manner, for example, by treatment with an appropriate base. An ester or salt of a compound of formula (1) may be converted into the parent compound, for example, by hydrolysis.
For a better understanding of the invention, the following Examples are given by way of illustration.